We present a protocol to create cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) for the optical detection of volumetric neurotransmitter release.
Cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) provide a new tool for neuroscientists to optically detect the release of neurotransmitters in the brain in vivo. A specific CNiFER is created from a human embryonic kidney cell that stably expresses a specific G protein-coupled receptor, which couples to Gq/11 G proteins, and a FRET-based Ca2+-detector, TN-XXL. Activation of the receptor leads to an increase in the FRET signal. CNiFERs have nM sensitivity and a temporal response of seconds because a CNiFER clone utilizes the native receptor for a particular neurotransmitter, e.g., D2R for dopamine. CNiFERs are directly implanted into the brain, enabling them to sense neurotransmitter release with a spatial resolution of less than one hundred µm, making them ideal to measure volume transmission in vivo. CNiFERs can also be used to screen other drugs for potential cross-reactivity in vivo. We recently expanded the family of CNiFERs to include GPCRs that couple to Gi/o G proteins. CNiFERs are available for detecting acetylcholine (ACh), dopamine (DA) and norepinephrine (NE). Given that any GPCR can be used to create a novel CNiFER and that there are approximately 800 GPCRs in the human genome, we describe here the general procedure to design, realize, and test any type of CNiFER.
To fully understand how neurons communicate in the brain, it is necessary to have a method to measure the release of neurotransmitters in vivo. There are several well-established techniques for measuring neurotransmitters in vivo. One commonly used technique is microdialysis, in which a cannula is inserted into the brain and a small volume of cerebrospinal fluid is collected and analyzed using high-performance liquid chromatography and electrochemical detection1. Microdialysis has a spatial resolution on the order of a few diameters of the probe, e.g., ~0.5 mm for a 200 μm diameter microprobe. The temporal resolution of this technique, however, is slow due to sampling intervals that typically last ~5 min or longer1. Moreover, analyses are not made in real-time. Another technique is fast scanning cyclic voltammetry (FSCV), which uses a carbon-fiber probe that is inserted into the brain. FSCV has excellent temporal resolution (subsecond), high sensitivity (nanomolar), and spatial resolution with probe diameters of 5 to 30 μm. However, FSCV is limited to transmitters that produce a characteristic oxidation and reduction profile with voltage on a carbon potentiometric probe2.
A third technique to measure neurotransmitters is directly through genetically-encoded neurotransmitter (NT) biosensors3. With this method, a fusion protein is created that contains a ligand-binding domain for a transmitter coupled to a fluorescence resonance energy transfer (FRET)-based pair of fluorophores4 or a permutated GFP5. Unlike the previous two methods, these biosensors are genetically encoded and expressed on the surface of a host cell, such as a neuron, through the production of transgenic animals or acutely with the use of viral agents to infect cells. To date, genetically-encoded biosensors have been only developed for detecting glutamate and GABA3-5. Limitations with these techniques have been the low sensitivity, in the nM range, and the inability to expand the detection to the large number of transmitters, e.g., classical neurotransmitters, neuropeptides and neuromodulators, which signal through G protein-coupled receptors (GPCRs). In fact, there are nearly 800 GPCRs in the human genome.
To address these shortfalls, we have developed an innovative tool to optically measure release of any neurotransmitter that signals through a GPCR. CNiFERs (cell-based neurotransmitter fluorescent engineered reporters) are clonal HEK293 cells engineered to express a specific GPCR that, when stimulated, triggers an increase in intracellular [Ca2+] that is detected by a genetically encoded FRET-based Ca2+ sensor, TN-XXL. Thus, CNiFERs transform neurotransmitter receptor binding into a change in fluorescence, providing a direct and real-time optical read-out of local neurotransmitter activity. By utilizing the native receptor for a given neurotransmitter, CNiFERs retain the chemical specificity, affinity and temporal dynamics of the endogenously expressed receptors. To date, we have created three types of CNiFERs, one for detecting acetylcholine using the M1 receptor, one for detecting dopamine using the D2 receptor, and one for detecting norepinephrine using the α1a receptor6,7. The CNiFER technology is readily expandable and scalable, making it amenable to any type of GPCR. In this JoVE article, we describe and illustrate the methodology to design, realize, and test in vivo CNiFERs for any application.
All animal procedures performed in this study are in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines, and have been approved by the IACUCs at the Icahn School of Medicine at Mount Sinai and the University of California, San Diego.
1. Generate GPCR-expressing Lentivirus for Transforming HEK293 Cells
2. Choosing HEK293/TN-XXL Backbone Cell Type for Culturing In Vitro
Note: Determine the G protein coupling specificity, e.g., Gi/o, Gq/11, or Gs G proteins, of the GPCR, as this dictates whether a G protein chimera is needed for the CNiFER. For Gq-coupled receptors, e.g., M1 muscarinic receptor, choose HEK293/TN-XXL(#3g8) as the backbone HEK293 cell type. For Gi/o-coupled receptors, the chimeric G protein Gqi5 is needed10. For Gs-coupled receptor, the Gqs5 chimera is needed10. In this protocol, the construction of a D2R CNiFER is used as an example. D2R signals through Gi/o G proteins and requires HEK293 cells that stably express the chimeric G protein, Gqi5, e.g., HEK293/TN-XXL/Gqi5_#qi5.6.
3. Lentiviral Transduction of HEK293/TN-XXL/Gqi5 Cells
4. FACS and Isolation of Single CNiFER Clones
5. Culturing and Expansion of Sorted, Clonal CNiFERs
6. Identify Candidate CNiFERs Based on FRET Response Using Fluorometric Plate Reader
Note: With four 96-well plates following FACS, there should be >100 testable clones that survive and expand to the 24-well plate stage, since many of the original clones fail to grow. To identify potential candidate CNiFERs, use a 3-point analysis for the FRET response with cognate agonist, e.g., dopamine for D2R.
7. Final Selection of CNiFER Clones Using Fluorometric Plate Reader
8. Freeze-back Selected CNiFER Clones
9. CNiFER Implantation into Mouse Cortex
10. In Vivo Imaging of CNiFER Clones
Note: Live imaging is conducted with mice using a two-photon microscope and a head-fixed apparatus. No anesthesia is needed during the imaging sessions. When imaging animals in the awake state, limit head restraint to only a few hours at a time to reduce stress levels. Return the animal to it home cage between imaging sessions for food and water. Potential stress is minimized by darkening the room lights and surrounding part of the mouse in an enclosure.
11. Data Analysis
A CNiFER is derived from a human embryonic kidney (HEK293) cell that is engineered to stably express at least two proteins: a specific G-protein coupled receptor (GPCR) and a genetically encoded [Ca2+] sensor, TN-XXL. TN-XXL undergoes fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent proteins, eCFP and Citrine, respectively, in response to Ca2+ ions6,15. Activation of GPCRs that couple to endogenous Gq G proteins trigger an increase in cytosolic [Ca2+] through the PLC/IP3 pathway, leading to an increase in FRET from the TNXXL Ca2+ detector (Figure 1).
Figure 1: Scheme for Developing CNiFERs. Top, GPCR-Ca2+ signaling pathway required for creating a CNiFER cell. Bottom, the basic steps for constructing CNiFERs using HEK293 cells. Step 1. Transduce with genetically encoded FRET-based Ca2+-detector (TN-XXL). Step 2. Transduce Gα G-protein chimera, i.e., Gqs5, Gqi5, if necessary. Step 3. Transduce unique GPCR to create CNiFER. Two-photon excitation light (red) excites eCFP, which undergoes FRET, producing both an eCFP emission (cyan) and Citrine emission (yellow). Please click here to view a larger version of this figure.
The increase in FRET provides a rapid optical read-out of the change in neurotransmitter levels. To develop a CNiFER for a particular type of neurotransmitter, first determine the type of G protein that couples to the GPCR. For Gq-coupled GPCRs, the GPCR uses Gq proteins endogenously expressed in HEK293 cells. For Gi/o-coupled GPCRs, a clonal HEK293 line is first created that expresses a chimeric G protein that redirects the GPCR to the Gq-PLC/IP3 pathway. This is accomplished with a chimeric G protein, Gqi5, which contains primarily Gαq sequence and five amino acids of the carboxyl terminus of Gi. These five amino acids are sufficient for Gqi5 to communicate with Gi/o-coupled GPCRs, but signal through the Gq pathway. For Gs-coupled GPCRs, a Gqs5 chimera is used10. The general strategy for producing a CNiFER is to: 1) create a clonal HEK293 cell that is stably expressing an optical Ca2+ detector, i.e., TN-XXL, using a lentivirus transduction of HEK cells, 2) stably express a G protein chimera, if necessary, in the HEK293 cell clone expressing TN-XXL, and 3) create a stably expressing GPCR clone in the HEK293 cell clone expressing TN-XXL and the chimeric G protein. The clonal HEK293 line that lacks the GPCR but has the TN-XXL and chimeric G protein serves as the 'control CNiFER'. The control CNiFER is needed to confirm that the CNiFER response is due specifically to activation of the engineered receptors, i.e., D2R, and not to activation of other receptors endogenously expressed in HEK293 cells.
To generate lentivirus, a lentivector expression system is used, e.g., pCDH-CMV-MCS-EF1-Puro, which contains the genetic elements responsible for packaging, transduction, stable integration of the viral expression construct into genomic DNA, and expression of the target gene sequence. To produce a high titer of viral particles, expression and packaging vectors are transiently co-transfected into producer mammalian cells and virus is collected. There are several viral core facilities in the US that can generate high titer lentivirus. Following infection of HEK293 cells, the Puro gene provides antibiotic resistance for identifying transduced HEK293 cells.
In order to identify specific clonal lines, transduced HEK293 cells are sorted using a fluorescence activated cell sorting (FACS) system. The objective is to isolate a clone that contains a high expression level of FRET-based Ca2+ detector and the capability of undergoing FRET. In this example of FACS analysis, the fluorescence of the eCFP emission is plotted against the FRET signal (eCFP excitation and Citrine emission). The boxes mark regions (P2 and P3) that will be subsequently selected ("gated") for sorting into 96-well plates (Figure 2). Generally, about four 96-well plates are sufficient to screen for successful creation of CNiFERs. From these 4 plates, approximately 100 clones are suitable for fluorometric plate reader analysis.
Figure 2: Example of FACS Analysis. A sample of the output following a FACS analysis. The graph plots eCFP emission ("475/20-A") as a function of Citrine emission ("FRET V-530/30-A"), using eCFP excitation for each cell. Regions P2 and P3 show areas selected, i.e., gated, for sorting into individual cells. Colors are arbitrary. Please click here to view a larger version of this figure.
Once the sorted cells have grown to sufficient density, the FRET response following agonist activation is determined using a 96-well fluorometric plate reader system equipped with solution handling. To narrow down the number of clones to study, a "3-point" agonist curve is used to screen ~100 clones and select CNiFERs with the best responses. Approximately 10 clones are then analyzed further with determination of the complete dose-response with the cognate agonist, and the non-specific responses, probed with 12 other neurotransmitters or modulators. A 96-well drug plate is prepared as three-fold concentration (final concentration is diluted 1:3 in plate) of drugs (e.g., agonists, antagonists, etc.) in ACSF. In this example, a drug plate is set up for testing a D2 CNiFER with its cognate agonist, dopamine, and potential non-specific responses with a variety of other neurotransmitter and peptide agonists (Figure 3). The backbone CNiFER, which lacks the GPCR, serves as an important control for the newly created CNiFER.
Figure 3: Examples of Layout for 96-well Plates. Top, table of the layout for loading a 3x drug plate for fluorometric plate reader, using three-fold concentrations of various neurotransmitters and peptides. Bottom, examples of clear plastic 96-well drug plate and black 96-well plate for seeding CNiFERs and measuring in plate reader. Please click here to view a larger version of this figure.
Stimulation of the GPCR is expected to increase the FRET response, as a consequence of an elevation of intracellular [Ca2+] and detection by TN-XXL. Under these conditions, FRET is produced by eCFP and Citrine moving closer, so that excitation of eCFP produces a smaller eCFP emission and larger Citrine emission. In this example, excitation is set to 436 nm and emission filters are set to 485 ± 7.5 nm for eCFP and 527 ± 7.5 nm for Citrine (Figure 4). Thirty sec of baseline fluorescence is measured and then 50 µl from the "three-fold" agonist in ACSF plate is delivered to each well containing 100 μl ACSF (1:3 dilution). eCFP and Citrine emission fluorescence are measured every 3.8 seconds for 180 sec. Background measurements are taken from wells without cells and subtracted, if necessary. Fluorescence intensities are normalized to pre-stimulus baselines (F(t)/F(baseline)), and peak responses are measured to calculate the FRET ratio (ΔR/R) using the F(t)/F(baseline) of the 527 nm and 485 nm channels (Equation 1). A dose response curve is then constructed by plotting the FRET ratio as a function of different agonist concentrations and fit with the Hill equation to determine the EC50 and Hill coefficient (Figure 5) (Equation 2). An optimal CNiFER exhibits a large FRET ratio and an appropriate EC50 for the cognate agonist, and exhibits little or no background responses to other neurotransmitter agonists. By contrast, the control CNiFER should show little response to the cognate agonist.
Figure 4: Example of Agonist-induced FRET Response. D2R CNiFER FRET response measured on a plate reader with a solution delivery system. (A) A plot of the FRET response, i.e., eCFP excitation with eCFP and Citrine emissions, during application of dopamine (red bar) to D2 CNiFERs. Note that eCFP emission decreases while Citrine emission increases with agonist (dopamine). (B) A plot of the FRET ratio (Equation 1) for the response in (A) Figure modified from Muller et al., 20147. Please click here to view a larger version of this figure.
Figure 5: Examples of Dose Response Curves for D2 CNiFER. (A) Dose response curves for response of D2 CNiFERs to dopamine (DA, black) and for norepinephrine (NE, green). In addition, the response of "control" CNiFERs lacking the D2R is shown. (B) The bar graph shows the FRET ratio response for other neurotransmitters and modulators at 50 nM and 1 μM. Values are mean ± SEM. Figure modified from Muller et al., 20147. Please click here to view a larger version of this figure.
CNiFER clones can be assessed further for possible receptor-dependent desensitization and for their temporal resolution, discriminating the presentation of two different agonist pulses (for details, see Muller et al., 20147). Having constructed a CNiFER clone, the next step is to test its function in vivo. To monitor the fluorescence in vivo, it is necessary to use a two-photon microscope. After preparing a thinned-skull window, CNiFERs are loaded into a glass pipette and injected into layers 2/3 of cortex. The mouse is then prepared for in vivo imaging by attaching a glass cover slip to the thinned skull, and implanting a head bar for fixing the head during imaging (Figure 6).
To determine that the implanted CNiFERs are viable in vivo, known concentrations of agonist can be injected near the site of implantation and the FRET ratio determined7. To further validate the activity of implanted CNiFERs, stimulating the input neurons should be examined. For example, with the D2 CNiFER, the effect of electrical stimulation of the midbrain dopamine neurons that project to the cortex was examined. A 0.1 MΩ tungsten bipolar stimulating electrode with a tip separation of 500 μm was implanted into the substantia nigra (−3.2 mm A/P, −1.3 mm M/L, −4.4 mm D/V). Figure 6 shows an example of electrically stimulating the substantia nigra at different intensities and observing an increase in the FRET ratio for D2 CNiFERs7. Note that systemic intra-peritoneal (i.p.) injection of a D2 receptor antagonist, eticlopride (1 mg/kg), blocks the D2 CNiFER response. On the other hand, injection of cocaine (15 mg/kg), which blocks reuptake of dopamine, enhances the electrically evoked D2 CNiFER response7.
Figure 6: Example of D2 CNiFER Response In Vivo Following Electrical Stimulation of Substantia Nigra. (A) A cartoon shows a head-fixed mouse prepared for in vivo two-photon imaging and electrical stimulation. Two-photon light (red, 820 nm) for excitation and 475 nm emission for eCFP (blue) and 530 nm emission for Citrine (green). (B) The line plot shows the FRET ratio for D2 CNiFER injected into cortex following electrical stimulation of substantia nigra, i.e., 200 μsec pulses of 50 to 300 μA at 50 Hz for 500 msec, and following electrical stimulation in the presence of a D2R antagonist (eticlopride) or cocaine. Figure modified from Muller et al., 20147. Please click here to view a larger version of this figure.
Table 1: List of chemicals and reagents for making HEK293 growth medium and ACSF.
Table 2: Volumes for Harvesting Cells From Different Size Culture Plates or Flasks.
The creation of CNiFERs provides an innovative and unique strategy for optically measuring release of neurotransmitters in the brain in vivo. CNiFERs are ideally suited for measuring extrasynaptic release, i.e., volume conduction, for neurotransmitters. Importantly, each CNiFER possesses the properties of the native GPCR, providing a physiological optical measurement of the changes in levels of neurotransmitters in the brain. To date, CNiFERs have been created for detecting acetylcholine (M1-CNiFER)6, dopamine (D2-CNiFER)7 and norepinephrine (α1a-CNiFER)7.
In principal, a CNiFER can be created for any neurotransmitter that signals through a GPCR. For the case where the GPCR signals through Gq G proteins, no further modification is needed to the HEK293 cell. GPCRs that signal through Gi/o, however, require coexpression of a Gqi5 chimeric G protein to couple the GPCR to the Gq/PLC pathway7,10. Similarly, GPCRs that signal through Gs will require coexpression of a chimeric Gqs5 G protein10. Once completed, each CNiFER clone is screened and only those CNiFER clones that have an affinity comparable to the native receptor, exhibit little or no desensitization and provide a signal-to-noise ratio that is adequate for measuring with in vivo two-photon microscopy, are selected for in vivo studies.
For in vivo studies, it is highly recommended to treat the mice with cyclosporine to minimize any potential immunological response. There is a possibility of rejection or an immunological response with implanting human CNiFER cells into the rodent brain. This was investigated previously by examining expression of GFAP and MAC17, following CNiFER implantation. CNiFERs did not appear to produce glial scars or generate any significant MAC1 staining7.
Two major issues to consider in constructing CNiFERs are sensitivity and desensitization. If the EC50 is too high, i.e., low affinity, relative to the native receptor, then the CNiFER may not have sufficient sensitivity to detect release of neurotransmitter in vivo. One solution is to rescreen clones and choose a different CNiFER clone that has higher affinity. An alternative strategy would be to test other types of genetically encoded fluorescent Ca2+-detectors that may have a higher Ca2+ sensitivity, which can shift the EC50 for GPCR activation. Because the CNiFER design is modular, it is easily adapted to other types of genetically encoded Ca2+-detectors, such as GCaMP16. Isolating CNiFER clones with the same receptor but different EC50s could be advantageous for extending the dynamic range of detecting release of endogenous neurotransmitters in vivo.
Desensitization of the CNiFER will also limit its use in vivo. If the peak response gradually decreases with each pulse of agonist, then the receptor may be desensitizing. In this case, examine other clones and determine if they respond the same way. Modifications to the receptor amino acid sequence, or use of another subtype of receptor may be necessary to address the agonist-dependent desensitization. If there are known sites of phosphorylation or amino acids identified that associate with G protein receptor kinases (GRKs), it would be advisable to construct a non-desensitizing variant of the GPCR by mutating one or more sites. The mechanism of desensitization must be determined for each receptor on a case-by-case basis.
Thus far, CNiFERs have been only implanted into superficial layers of cortex6,7, due to spectroscopic limitations with imaging fluorophores with two-photon microscopy17,18. In the future, it may be possible to adapt CNiFER technology with fiber-based measurements of fluorescence19 so that CNiFERs can be implanted in subcortical brain regions.
The authors have nothing to disclose.
We thank B. Conklin (University of California, San Francisco) for providing the Gqi5 and Gqs5 cDNAs, A. Schweitzer for assistance with the electronics, N. Taylor for assistance with screening of clones, Ian Glaaser and Robert Rifkin for proof reading, and Olivier Griesbeck for TN-XXL. This work was supported by research grants through the US National Institute on Drug Abuse (NIDA) (DA029706; DA037170), the National Institute of Biomedical Imaging and Bioengineering (NIBIB) (EB003832), Hoffman-La Roche (88610A) and the "Neuroscience Related to Drugs of Abuse" training grant through NIDA (DA007315).
pCDH-CMV-MCS-EF1-Puro | System Biosciences | CD510B-1 | Cloning: for generating lentivirus |
12×75 *BD Falcon High Clarity Polypropylene Round Bottom Test Tube | BD Biosciences | 352063 | FACS |
BD 40 um Falcon cell strainers | BD Biosciences | 352340 | FACS |
0.05% Trypsin EDTA | Invitrogen | 25200056 | FACS |
96 Well Plate, flat bottom, clear | Corning | 3596 | FACS |
96 well cell culture plates | Corning | CLS3997 | Flexstation |
Optilux black clear bottom | Corning | 3603 | Flexstation |
Flexstation pipet tips | Molecular Devices | 9000-0911 | Flexstation |
Acetylcholine Chloride | SimgaAldrich | A2661 | Flexstation |
Norepinephrine | SimgaAldrich | A7256 | Flexstation |
Dopamine Hydrochloride | SimgaAldrich | PHR1090 | Flexstation |
GABA | SimgaAldrich | A2129 | Flexstation |
Histamine | SimgaAldrich | H7125 | Flexstation |
Glutamate | SimgaAldrich | 49621 | Flexstation |
Epinephrine | SimgaAldrich | E4642 | Flexstation |
Somatostatin | SimgaAldrich | S1763 | Flexstation |
5HT | SimgaAldrich | H9523 | Flexstation |
VIP | Alpha Diagnostics Inc. | SP-69627 | Flexstation |
Orexin A | Alpha Diagnostics Inc. | 12-p-01 | Flexstation |
Substance P | SimgaAldrich | S6883 | Flexstation |
Adenosine | SimgaAldrich | A4036 | Flexstation |
Melatonin | SimgaAldrich | M5250C | Flexstation |
Fluorescence Plate Reader & software | Molecular Devices | Flexstation 3 | Flexstation |
DMEM (high glucose) with Glutamax | Life Technologies | 10569-010 | Tissue culture |
Fetal bovine serum | Life Technologies | 10082-139 | Tissue culture |
Pen/Strep | Life Technologies | 15140-122 | Tissue culture |
Puromycin | InvivoGen | ant-pr-1 | Tissue culture |
Fibronectin | SimgaAldrich | F0895 | Tissue culture |
CoolCell LX Alcohol-free controlled-rate cell freezing box | Bioexpress | D-3508) | Tissue culture |
cyanoacrylate glue | Loctite | Loctite no. 495 | surgery and stereotaxic injection |
plastic paraffin film | VWR | Parafilm® | surgery and stereotaxic injection |
NANOINJECTOR | Drummond | 3-000-204 | surgery and stereotaxic injection |
GLASS ELECTRODES | Drummond | 3-000-203G | surgery and stereotaxic injection |
hand held drill | OSADA | Exl-M40 | surgery and stereotaxic injection |
Burrs for drill | Fine Scientific | 19007-05; 19007-07) | surgery and stereotaxic injection |
Sterilizing bath | FST | 18000-45, Hot Bead Sterilizer | surgery and stereotaxic injection |
isoflurane chamber/mask | Highland Medical Equipment | 564-0427, HME 109 Table Top Anesthetic Machine with Isoflurane Vaporizer, O2 Flowmeter, Gang Valve; 564-0852, Induction Chamber 16X7X7.5cm | surgery and stereotaxic injection |
3D scope with arm | Zeiss | surgery and stereotaxic injection | |
fiber optic light | surgery and stereotaxic injection | ||
Betadine | surgery and stereotaxic injection | ||
70 % (v/v) isopropyl alcohol | surgery and stereotaxic injection | ||
Povidone-Iodine Prep Pads | dynarex | 1108 | surgery and stereotaxic injection |
NaCl 0.9% (INJECTION, USP, 918610) | surgery and stereotaxic injection | ||
CYCLOSPORINE (INJECTION, USP) | surgery and stereotaxic injection | ||
Buprenex (INJECTION) buprenorphine (0.03 μg per g rodent) | Sigma | surgery and stereotaxic injection | |
Ophthalmic ointment | Akorn | NDC 17478-235-35 | surgery and stereotaxic injection |
Surgifoam | Ethicon | surgery and stereotaxic injection | |
Grip dental cement | Dentsply | #675571, 675572 | surgery and stereotaxic injection |
Instant SuperGlue | NDindustries | surgery and stereotaxic injection | |
LOCTITE 4041 | surgery and stereotaxic injection | ||
METABOND | C&B | surgery and stereotaxic injection | |
no. 0 cover glass | Fisher | surgery and stereotaxic injection | |
stereotaxic frame | Kopf | surgery and stereotaxic injection | |
Rectal probe and heating pad | FHC | 40-90-8D, DC Temperature Controller,40-90-2-06, 6.5X9.5cm Heating Pad40-90-5D-02, Rectal Thermistor Probe | surgery and stereotaxic injection |
optical breadboard for imaging | Thorlabs | surgery and stereotaxic injection | |
Mineral oil | Fisher | S55667 | surgery and stereotaxic injection |
Kwik-Cast (Silicone elastomer) | World Precision Instruments | surgery and stereotaxic injection | |
Suture | Ethicon | 18’’, 1667, 4-0 | surgery and stereotaxic injection |
Scissors | Fine Scientific Tools | 91500-09, 15018-10 | surgery and stereotaxic injection |
Forcepts | Fine Scientific Tools | 11252-30; #55, 11295-51; Grafe, 11050-10 | surgery and stereotaxic injection |
Student Halsted-Mosquito Hemostats | Fine Scientific Tools | 91308-12 | surgery and stereotaxic injection |
Small Vessel Cauterizer Kit | Fine Scientific Tools | 18000-00 | surgery and stereotaxic injection |
Hot Bead Sterilizers | Fine Scientific Tools | 18000-45 | surgery and stereotaxic injection |
Instrument Case with Silicone Mat | Fine Scientific Tools | 20311-21 | surgery and stereotaxic injection |
Plastic Sterilization Containers with Silicone Mat | Fine Scientific Tools | 20810-01 | surgery and stereotaxic injection |
2P fixed-stage fluorescence scope for in vivo imaging | Olympus | FV1200 MPE | in vivo imaging |
Multiphoton laser | SpectraPhysics | Mai Tai DeepSee | in vivo imaging |
Green Laser | Olympus | 473 nm Laser | in vivo imaging |
xy translation base | Scientifica | MMBP | in vivo imaging |
FRET filter cube for YFP and CFP | Olympus | in vivo imaging | |
25-X water immersion objective | Olympus | in vivo imaging | |
air table | Newport | in vivo imaging | |
custom built light-tight cage | Thorlab | in vivo imaging |